CN101454345B - High affinity antibodies to human il-6 receptor - Google Patents

Abstract

The present application provides human antibodies or antigen-binding fragments thereof, K that bind to the human IL-6 receptor (hIL-6R)_DAbout 500pM or less, blocks the IC of IL-6 activity₅₀200pM or less. The antibody or antibody fragment binds hIL-6R with at least two-fold greater affinity than monkey IL-6R.

Description

High affinity antibody to human IL-6 receptor

Description of related fields

Interleukin-6 (IL-6), a pleiotropic cytokine produced by immune and non-immune cells, plays a crucial role in the regulation of immune responses, acute phase responses, and hematopoiesis. It binds to soluble and membrane-bound IL-6R (α-chain) to form a binary complex that can interact with membrane-bound gp130 (β-chain) to induce the formation of a signaling complex, which Contains two each of IL-6, IL-6R and gp130.

Antibodies to hIL-6R are described in US 5,670,373, 5,795,965, 5,817,790, 6,410,691 and EP 409 607B1. Methods of treatment are described in US 5,888,510 and 6,723,319.

Summary of the invention

In the first aspect, the present invention provides a human antibody, preferably a recombinant human antibody, that specifically binds to human interleukin-6 receptor (hIL-6R). These antibodies are characterized by binding to hIL-6R with high affinity and low dissociation kinetics and are able to neutralize the activity of IL-6. Antibodies can be full length (e.g., IgG1 or IgG4 antibodies), or may comprise only an antigen-binding portion (e.g., Fab, F(ab') ₂ , or scFv fragments), possibly modified to affect functionality, e.g., to eliminate residual Effector function (Reddy et al. (2000) J. Immunol. 164:1925-1933). In a preferred embodiment, the invention provides antibodies or antigen-binding fragments thereof that bind to human IL _- 6 receptor (SEQ ID NO: 1) with a _KD of about 500 pM or less, as measured by surface plasmon resonance. In more specific embodiments, the antibody or antigen-binding fragment has a _KD of less than 300 pM, or less than 200 pM, or even less than 100 pM. In various embodiments, the antibody or antigen-binding fragment _thereof blocks hIL-6 activity with an _IC50 of 250 pM or less, as measured using a luciferase bioassay. In a more specific embodiment, the antibody or antigen-binding fragment thereof exhibits an _IC50 of 150 pM or less.

In a related aspect, an antibody or antigen-binding fragment of the invention binds hIL-6R with at least two-fold higher affinity than it binds monkey IL-6R. In a more preferred embodiment, the antibody or antigen-binding fragment binds to hIL-6R protein (SEQ ID NO: 1) more than it binds to monkey IL-6R (Macaca fascicularis) ectodomain, such as SEQ ID NO: 251) binds with approximately three-fold higher affinity.

In one embodiment, an antibody or antigen binding portion of an antibody of the invention comprises a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO: 3, 227, 19, 231, 35, 51, 67, 83, 99, 115 , 131, 147, 239, 241, 163, 179, 235, 195 and 211, or sequences substantially similar to these. In a more specific embodiment, the antibody or antigen-binding fragment thereof further comprises a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 11, 229, 27, 233, 43, 59, 75, 91, 107, 123 , 139, 155, 171, 187, 237, 203 and 219, or sequences substantially similar to these. In specific embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR/LCVR pair selected from the group consisting of SEQ ID NO: 3/11; 227/229; 19/27; 231/233; 35/43; 51/59; 67/ 75; 83/91; 99/107; 115/123; 131/139; 147/155; 239/155; 241/155; 163/171; 179/187; 235/237; , or sequences substantially similar to these.

In a second aspect, the invention provides an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment of an antibody of the invention. In one embodiment, the nucleic acid molecule of the present invention encodes an antibody or fragment thereof comprising the HCVR described above. In particular embodiments, the nucleic acid molecule encoding HCVR is selected from the group consisting of SEQ ID NO: 2, 226, 18, 230, 34, 50, 66, 82, 98, 114, 130, 146, 238, 240, 162, 178, 234 , 194 and 210, or sequences substantially identical to these. In a related aspect, the invention provides isolated nucleic acid molecules encoding the LCVRs described above. In a particular embodiment, the nucleic acid molecule encoding LCVR is a nucleotide sequence selected from the group consisting of SEQ ID NO: 10, 228, 26, 232, 42, 58, 74, 90, 106, 122, 138, 154, 170 , 186, 236, 202 and 218, or sequences substantially identical to these.

In a third aspect, the invention provides an antibody or antigen-binding fragment comprising a heavy chain complementarity determining region 3 (CDR3) domain and a light chain CDR3 domain, wherein

The heavy chain CDR3 domain comprises an amino acid sequence of the following formula: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ -X ¹² -X ¹³ -X ¹⁴ -X ¹⁵ -X ¹⁶ -X ¹⁷ -X ¹⁸ -X ¹⁹ (SEQ ID NO: 247), wherein X ¹ =Ala, X ² =Lys, X ³ =Gly, X ⁴ =Arg, X ⁵ =Asp , X ⁶ =Ser or Ala, X ⁷ =Phe, X ⁸ =Asp; X ⁹ =Ile, X ¹⁰ =Pro or deletion, X ¹¹ =Phe or deletion, X ¹² =Val or deletion, X ¹³ =Tyr or deletion , X ¹⁴ =Tyr or deletion, X ¹⁵ =Tyr or deletion, X ¹⁶ =Gly or deletion, X ¹⁷ =Met or deletion, X ¹⁸ =Asp or deletion, X ¹⁹ =Val or deletion; and

The light chain CDR3 domain comprises an amino acid sequence of the following formula: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ (SEQ ID NO: 250), wherein X ¹ = Gln, X ² =Gln or His, X ³ =Ala, X ⁴ =Asn or Tyr, X ⁵ =Ser, X ⁶ =Phe, X ⁷ =Pro, X ⁸ =Pro, X ⁹ =Thr.

In a more specific embodiment, the antibody or antigen-binding fragment further comprises

A heavy chain CDR1 domain comprising an amino acid sequence of the formula: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ (SEQ ID NO: 245), wherein X ¹ =Gly or Arg, X ² =Phe, X ³ =Thr, X ⁴ =Phe, X ⁵ =Asp, X ⁶ =Asp, X ⁷ =Tyr, X ⁸ =Ala;

A heavy chain CDR2 domain comprising an amino acid sequence of the following formula: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ (SEQ ID NO: 246), wherein X ¹ =Ile or Val, X ² =Ser, X ³ =Trp, X ⁴ =Asn, X ^s =Ser, X ⁶ =Gly, X ⁷ =Ser, X ⁸ =Ile;

A light chain CDR1 domain comprising an amino acid sequence of the following formula: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ (SEQ ID NO: 248), wherein X ¹ =Gln , X ² =Gly, X ³ =Ile, X ⁴ =Ser, X ⁵ =Ser, X ⁶ =Trp; and

A light chain CDR2 domain comprising an amino acid sequence of the following formula: X ¹ -X ² -X ³ (SEQ ID NO: 249), wherein X ¹ =Gly or Ala, X ² =Ala, X ³ =Ser.

In a fourth aspect, the present invention provides an antibody or antigen-binding fragment comprising:

A heavy chain CDR3 domain selected from the group consisting of SEQ ID NOS: 25, 153, 9, 185, 41, 57, 73, 89, 105, 121, 137, 169, 201 and 217; and

A light chain CDR3 domain selected from the group consisting of SEQ ID NO: 33, 161, 17, 193, 49, 65, 81, 97, 113, 129, 145, 177, 209 and 225.

In a more specific embodiment, the antibody or antigen-binding fragment further comprises:

a heavy chain CDR1 domain selected from the group consisting of SEQ ID NOS: 21, 149, 5, 181, 37, 53, 69, 85, 101, 117, 133, 165, 197, and 213;

a heavy chain CDR2 domain selected from the group consisting of SEQ ID NOS: 23, 151, 7, 183, 39, 55, 71, 87, 103, 119, 135, 167, 199, and 215;

A light chain CDR1 domain selected from the group consisting of SEQ ID NOS: 29, 157, 13, 189, 45, 61, 77, 93, 109, 125, 141, 173, 205, and 221; and

A light chain CDR2 domain selected from the group consisting of SEQ ID NOS: 31, 159, 15, 191, 47, 63, 79, 95, 111, 127, 143, 175, 207 and 223.

In specific embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR sequences SEQ ID NO: 21, 23, 25 and light chain CDR sequences SEQ ID NO: 29, 31, 33; heavy chain CDR sequences SEQ ID NO: 149, 151 , 153 and light chain CDR sequence SEQ ID NO: 157, 159, 161; heavy chain CDR sequence SEQ ID NO: 5, 7, 9 and light chain SEQ ID NO: 13, 15, 17; heavy chain CDR sequence SEQ ID NO : 181, 183, 185 and light chain CDR sequence SEQ ID NO: 189, 191, 193.

In a fifth aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention, wherein the antibody or fragment thereof comprises:

A heavy chain CDR3 domain encoding a nucleotide sequence selected from the group consisting of SEQ ID NO: 24, 152, 8, 184, 40, 56, 72, 88, 104, 120, 136, 168, 200 and 216; and

Light chain CDR3 structural domain, its coding nucleotide sequence is selected from SEQ ID NO:32,160,16,192,48,64,80,96,112,128,144,176,208 and 224; the same nucleic acid sequence.

In a more specific embodiment, there is provided an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment of the invention, wherein the antibody or fragment thereof comprises:

Heavy chain CDR1, its coding nucleotide sequence is selected from SEQ ID NO: 20, 148, 4, 180, 36, 52, 68, 84, 100, 116, 132, 164, 196 and 212;

A heavy chain CDR2 domain whose coding nucleotide sequence is selected from SEQ ID NO: 22, 150, 6, 182, 38, 54, 70, 86, 102, 118, 134, 166, 198 and 214;

A light chain CDR1 domain encoding a nucleotide sequence selected from the group consisting of SEQ ID NO: 28, 156, 12, 188, 44, 60, 76, 92, 108, 124, 140, 172, 204 and 220; and

A light chain CDR2 domain, which encodes a nucleotide sequence selected from the group consisting of SEQ ID NO: 30, 158, 14, 190, 30, 46, 62, 78, 94, 110, 126, 142, 174, 206 and 222; and These are substantially identical nucleic acid sequences.

The invention encompasses anti-hIL-6R antibodies or antigen-binding fragments thereof having modified glycosylation patterns. In some applications, antibodies that have been modified to remove undesired glycosylation sites or lack fucose moieties on the oligosaccharide chain can be used, for example, to increase antibody-dependent cell-mediated cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277:26733). Among other applications, galactosylation modifications can be made to alter complement dependent cytotoxicity (CDC).

In other aspects, the present invention provides a recombinant expression vector carrying the nucleic acid molecule of the present invention, a host cell into which the vector is introduced, and a method for producing the antibody or antigen-binding fragment of the present invention by culturing the host cell of the present invention. The host cells can be prokaryotic or eukaryotic cells, preferably the host cells are Escherichia coli (Ecoli) cells or mammalian cells, such as CHO cells.

In other aspects, the present invention provides a pharmaceutical composition comprising a human antibody or an antigen-binding fragment of an antibody that specifically binds hIL-6R and a pharmaceutically acceptable carrier.

In other aspects, the invention provides methods of inhibiting human IL-6 activity using the antibodies of the invention, or antigen-binding portions thereof. In one embodiment, the present invention includes a method of treatment comprising administering an antibody or fragment thereof of the present invention to a human subject whose condition is treated or ameliorated by inhibiting IL-6 activity. The disorder can be, for example, arthritis, including chronic rheumatoid arthritis; inflammatory bowel disease, including Crohn's disease and ulcerative colitis; systemic lupus erythematosus and inflammatory diseases.

In other aspects, the present invention provides the use of an antibody or an antigen-binding fragment of an antibody as defined above for the preparation of a medicament for alleviating or inhibiting an IL-6-mediated human disease or disorder. In a related aspect, the invention provides an antibody or an antigen-binding fragment of an antibody as defined above, for use in alleviating or inhibiting an IL-6 mediated disease or condition in a human.

Other objects and advantages of the present invention will become apparent from the following detailed description.

Detailed description of the invention

Before the methods of the present invention are described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not limiting, since the scope of the present invention will be defined only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

The term "human IL6R" (hIL-6R), as used herein, means a human cytokine receptor that specifically binds to interleukin-6 (IL-6). The extracellular domain of hIL-6R is represented by SEQ ID NO: 1.

The term "antibody", as used herein, means an immunoglobulin molecule comprising four polypeptide chains: two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion" of an antibody (or simply "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain specific binding to an antigen (e.g., hIL-6R) Ability. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) Fab fragments, monovalent fragments comprising VL, VH, CL1 and CH1 domains; (ii) F(ab') ₂ fragments, bivalent Fragment, comprising two Fab fragments, connected by a disulfide bond in the hinge region; (iii) Fd fragment, comprising VH and CH1 domains; (iv) Fv fragment, comprising VL and VH domains of a single arm of the antibody, (v) dAb fragments (Ward et al. (1989) Nature 241:544-546), comprising a VH domain; and (vi) isolated complementarity determining regions (CDRs). In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by recombinant means by making them into a single continuous chain through a synthetic linker, in which the VL and VH regions pair to form a monovalent molecule (called a monovalent molecule). Chain Fv (scFv); see eg, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The term "antigen-binding portion" of an antibody is also intended to encompass such single chain antibodies. Other forms of single chain antibodies are also contemplated, such as diabodies (see eg, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).

A "neutralizing" or "blocking" antibody as used herein means an antibody that binds to hIL-6R, resulting in inhibition of the biological activity of hIL-6. Response to hIL-6 can be assessed by measuring one or more indicators of hIL-6 biological activity known in the art, such as hIL-6-induced cell activation and binding of hIL-6 to hIL-6R (see Examples below). Inhibition of biological activity.

"CDRs" or complementarity determining regions are hypervariable regions interspersed with more conserved regions called "framework regions" (FR). In various embodiments of the anti-hIL-6R antibodies or fragments of the invention, the FRs may be identical to human germline sequences, or may be naturally or artificially modified. A set of CDRs can be defined as an amino acid consensus sequence; for example, in one embodiment, an anti-hIL-6R antibody or antigen-binding fragment of the invention can be described as comprising a heavy chain CDR3 domain comprising the amino acid sequence of the formula : X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ -X ¹² -X ¹³ -X ¹⁴ -X ¹⁵ -X ¹⁶ -X ¹⁷ -X ¹⁸ -X ¹⁹ (SEQ ID NO: 247), wherein X ¹ =Ala, X ² =Lys, X ³ =Gly, X ⁴ =Arg, X ⁵ =Asp, X ⁶ =Ser or Ala, X ⁷ =Phe, X ⁸ =Asp; X ⁹ =Ile, X ¹⁰ =Pro or deletion, X ¹¹ =Phe or deletion, X ¹² =Val or deletion, X ¹³ =Tyr or deletion, X ¹⁴ =Tyr or deletion, X ¹⁵ = Tyr or deletion, X ¹⁶ =Gly or deletion, X ¹⁷ =Met or deletion, X ¹⁸ =Asp or deletion, X ¹⁹ =Val or deletion; and a light chain CDR3 domain comprising an amino acid sequence of the formula: X ¹ -X ² -X ³ -X ⁴ -X ⁵ -X ⁶ -X ⁷ -X ⁸ -X ⁹ (SEQ ID NO: 250), wherein X ¹ =Gln, X ² =Gln or His, X ³ =Ala , X ⁴ =Asn or Tyr, X ⁵ =Ser, X ⁶ =Phe, X ⁷ =Pro, X ⁸ =Pro, X ⁹ =Thr.

The term "surface plasmon resonance" as used herein refers to an optical phenomenon, such as by detecting changes in protein concentration in a biosensor matrix using a BIAcore ^TM system (Pharmacia Biosensor AB, Pharmacia Biosensor Company), which allows real-time analysis of proteins interaction.

The term "epitope", is an antigenic determinant that interacts with a specific antigen-binding site called a paratope in the variable region of an antibody molecule. A single antigen may have more than one epitope. An epitope can be a conformational epitope or a linear epitope. Conformational epitopes result from the spatial juxtaposition of amino acids from different parts of the linear polypeptide chain. Linear epitopes arise from contiguous amino acid residues in a polypeptide chain. In some cases, an epitope may include a carbohydrate, phosphoryl or sulfonyl moiety on the antigen.

The term "substantial identity" or "substantially identical", when referring to a nucleic acid or fragment thereof, means that it is optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions , the nucleotide sequence identity in nucleotide bases is at least about 95%, more preferably at least about 96%, 97%, 98% or 99%, as determined using any well-known sequence identity algorithm, such as discussed below FASTA, BLAST or Gap.

When applied to polypeptides, the term "substantial similarity" or "essentially similar" means that two peptide sequences share at least 95% sequence identity when optimally aligned, for example with the GAP or BESTFIT programs, using default gap weights Even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is the substitution of an amino acid residue by another amino acid residue with a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). Typically, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be increased due to the conservative nature of the substitutions. Methods for making this adjustment are well known to those skilled in the art. See, eg, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains, cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid- aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., (1992) Science 256: 144345. "Moderate conservatism" was replaced by any change that had a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity, also known as sequence identity, of polypeptides is usually determined using sequence analysis software. Protein analysis software matches similar sequences using similarity measures that assign values to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used to determine sequence homology or sequence identity between closely related polypeptides with default parameters, such as homologous polypeptides from different species of organisms, or wild-type proteins and other proteins. mutant protein. See, eg, GCG Version 6.1. FASTA can also be used to compare polypeptide sequences with default or recommended parameters, GCG version 6.1. The programs in FASTA (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identities (Pearson (2000), op. cit.). Another preferred algorithm when comparing sequences of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly blastp or tblastn, using default parameters. See, e.g., Altschul et al., (1990) J. Mol. Biol. 215: 403 410 and Altschul et al., (1997) Nucleic Acids Res. 25: 3389 402.

Production of Human Antibodies

Methods for generating human antibodies include, for example, Veloclmmune ^™ (Regeneron Pharmaceuticals), XenoMouse ^™ technology (Green et al. (1994) Nature Genetics 7:13-21; Abgenix Corporation), "minilocus" Methods and phage display (see, eg, US 5,545,807, US 6,787,637). Veloclmmune ^™ technology (US 6,596,541 ) encompasses a method for generating highly specific fully human antibodies against selected antigens. This technique involves the creation of transgenic mice whose genomes contain human light chains and light chain variable regions operably linked to the mouse endogenous constant region loci, so that the mice respond to antigenic stimulation to produce mice containing human variable regions and light chain variable regions. Antibodies with constant regions. The DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to the DNA encoding the constant regions of the human heavy and light chains. The DNA is then expressed in cells capable of expressing fully human antibodies. In specific embodiments, the cells are CHO cells.

Antibodies can be used therapeutically to block ligand-receptor interactions or to inhibit receptor component interactions other than through complement fixation (complement-dependent cytotoxicity) (CDC) and participation in antibody-dependent cell-mediated cellular Cytotoxicity (ADCC) kills cells. The constant region of an antibody is important for the ability of the antibody to bind complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether the antibody is desired to mediate cytotoxicity.

Human immunoglobulins can exist in two forms associated with hinge region heterogeneity. In one form, immunoglobulin molecules comprise a stable four-chain structure, approximately 150-160 kDa, in which dimers are joined by heavy chain disulfide bonds between the chains. In the second form, the dimers are not linked by interchain disulfide bonds, forming a molecule of about 75-80 kDa comprising covalently coupled light and heavy chains (half antibodies). These forms are extremely difficult to isolate, even after affinity purification. The frequency with which the second form occurs among the various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody's hinge region isotype. In fact, single amino acid substitutions in the hinge region of human IgG4 can significantly reduce the appearance of the second form (Angal et al., (1993) Molecular Immunology 30:105), to levels normally observed when using the human IgG1 hinge. The invention encompasses antibodies having one or more mutations in the hinge, CH2 or CH3 regions, which may be desirable, for example, in manufacturing to increase yields of the desired antibody form.

Antibodies of the invention are preferably prepared using Veloclmmune( ^TM) technology. The heavy and light chain variable regions of endogenous immunoglobulins in transgenic mice are replaced with the corresponding human variable regions, such transgenic mice are challenged with the antigen of interest, and lymphocytes (such as B cell). Lymphocytes can be fused with myeloma cell lines to produce immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for an antigen of interest. The DNA encoding the heavy and light chain variable regions can be separated and joined to the heavy and light chain constant regions of the desired isotype. Such antibody proteins can be produced in cells, such as CHO. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable regions can be isolated directly from antigen-specific lymphocytes.

In one embodiment, the transgenic mouse comprises up to 18 functional human variable heavy chain genes and 12 functional human variable kappa light chain genes. In another embodiment, the transgenic mouse comprises up to 39 human variable heavy chain genes and 30 human variable kappa light chain genes. In yet another embodiment, the transgenic mouse comprises up to 80 human variable heavy chain genes and 40 human variable kappa light chain genes.

In general, the antibodies of the invention have very high affinity, typically having a _{KD of} about 10 ^"9 to about 10" ¹² M as determined by binding to antigen immobilized on a solid phase or in a liquid phase.

First, high-affinity chimeric antibodies with human variable regions and mouse constant regions are isolated. Antibodies with desired characteristics, including binding affinity for hIL-6R, ability to block hIL-6, and/or selectivity for human proteins, are characterized and selected as described below. The mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies of the invention, such as wild-type or modified IgG4 or IgG1 (e.g., SEQ ID NO: 242, 243, 244). The high affinity antigen-binding and target-specific features are located in the variable region, although the constant region chosen will vary according to the particular application.

Epitope Mapping and Related Techniques

To screen for antibodies that bind to a particular epitope, a conventional cross-blocking assay may be used, such as that described in Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory, Harlow and Lane eds. Other methods include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavage assays as described in the Examples below. In addition, methods such as epitope excision, epitope extraction, and antigen chemical modification (Tomer (2000) Protein Science: 9:487-496) can be used.

Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is based on the chemical or enzymatic modification of each antibody on the surface of the antigen (US Patent Application Publication No. 2004/0101920) A method for sorting large numbers of monoclonal antibodies (mAbs) directed against the same antigen by similarity in characteristics. Each class may reflect a unique epitope, distinct from or partially overlapping with an epitope represented by another class. This technique enables rapid filtering of genetically identical antibodies, allowing for focused characterization of genetically distinct antibodies. When used in hybridoma screening, MAP can help identify rare hybridoma clones with desired characteristics. MAP can be used to classify hIL-6R antibodies of the invention into groups of antibodies that bind different epitopes.

Substances that can be used to alter the structure of immobilized antigens include enzymes such as proteolytic enzymes and chemical reagents. Antigenic proteins can be immobilized on the surface of the biosensor chip or on polystyrene beads. The latter can be assayed, for example, with the multiplex Luminex ^™ detection assay (Luminex Corp., Texas ^, USA). With multiplexing capacity that can handle up to 100 different types of beads, Luminex ^™ offers a virtually unlimited number of antigen surfaces with multiple modifications, enabling antibody epitope typing with higher resolution than biosensor assays.

Therapeutic Administration and Dosage Forms

Therapeutic entities according to the invention are administered with suitable carriers, excipients and other agents incorporated into formulations to provide improved transfer, delivery, tolerance, etc. properties. Many suitable dosage forms can be found in a formulary known to all pharmacists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company), Easton, PA, USA, 1975 ), especially Chapter 87, by Blaug, Seymour. These dosage forms include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as Lipofectin ^™ ), DNA conjugates, anhydrous absorbed Pastes, oil-in-water emulsions and water-in-oil emulsions, carbowaxes (polyethylene glycols of various molecular weights) emulsifiers, semisolid gels, and semisolid mixtures of carbowaxes. Any of the aforementioned mixtures may be suitable for treatment and therapy according to the invention, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerated for the route of administration. For additional information on excipients and carriers well known to pharmacists, see also Powell et al., PDA (1998) J Pharm SciTechnol. 52:238-311 and references cited therein.

Example

The following examples are intended to provide a complete disclosure and illustration of how to make and use the methods and compositions of the present invention for those of ordinary skill in the art, but not to limit the scope of the invention as considered by the inventors. Efforts have been made to be precise with respect to numbers used (eg, amounts, temperature, etc.), but experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1. Production of Human Antibodies to Human IL-6 Receptor

Rodents can be immunized by any method known in the art (see, eg, Harlow and Lane (1988), supra; Malik and Lillehoj, Antibody techniques: Academic Press, 1994, California, USA). In a preferred embodiment, hIL-6R antigen and an adjuvant (Veloclmmune ^™ , Regina Rong Pharmaceutical Co. (Regeneron Pharmaceuticals, Inc.); US 6,596,541), to stimulate an immune response. Such adjuvants include complete and incomplete Freund's adjuvant, the MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptide) (see O'Hagan, Vaccine adjuvant, by Human Press, 2000, New Jersey, USA). Such adjuvants prevent the rapid spread of the polypeptide by sequestering the antigen in a local depot and may contain factors capable of stimulating an immune response in the host. In one embodiment, hIL-6R is administered indirectly using a DNA plasmid comprising the hIL-6R gene, which is expressed using the protein expression machinery of the host cell to produce the antigenic polypeptide in vivo. In both approaches, the immunization schedule requires several doses spaced several weeks apart. Antibody immune responses are monitored using standard antigen-specific immunoassays. When the animal's immune response is maximal, the antibody-expressing B cells are harvested and fused with mouse myeloma cells to maintain their viability to form hybridoma cells. To select monoclonal antibodies with desired functions, the conditioned media of hybridoma cells or transfected cells (described below) were screened based on specificity, antigen binding affinity, and ability to block hIL-6 binding to hIL-6R.

Example 2. Production of anti-hIL6R antibodies by direct isolation of splenocytes

DNA encoding the VH and VL domains can be isolated directly from B cells positive for a single antigen. Briefly, hIL-6Rα-immunized transgenic mice were sacrificed and splenocytes were harvested. Red blood cells were removed by lysis followed by sedimentation of harvested splenocytes. Resuspended splenocytes were first incubated for 1 hour with a mixture of human IgG, FITC-anti-mFc and biotin-IL6Ra. Stained cells were washed twice with PBS and then stained for 1 hour with a mixture of human and rat IgG, APC-anti-mIgM and SA-PE. The stained cells were washed once with PBS, and analyzed by flow cytometry using MoFlo (Cytomation). The IgG-positive, IgM-negative and antigen-positive B cells were sorted and put into different wells of a 96-well plate. Antibody genes from these B cells were subjected to RT-PCR according to the method described by Wang et al. (2000) (J Immunol Methods 244:217-225). Briefly, cDNA of each individual B cell was synthesized by RT-PCR. The resulting RT products were then divided into two and transferred to two corresponding wells on two 96-well plates. Using a 5' degenerate primer specific to the leader sequence of the human IgG heavy chain variable region and a 3' primer specific to the mouse heavy chain constant region, a set of obtained RT products was first amplified by PCR to form an amplicon. This amplicon was then amplified again by PCR using a 5' degenerate primer set specific for human IgG heavy chain variable region sequence framework 1 and a 3' nested primer specific for mouse heavy chain constant region. Using a 5' degenerate primer specific to the leader sequence of the variable region of the human κ light chain and a 3' primer specific to the constant region of the mouse κ light chain, a set of obtained RT products was first amplified by PCR to form amplicons. This amplicon was then amplified again by PCR using a 5' degenerate primer set specific for human kappa light chain variable region sequence framework 1 and a 3' nested primer specific for mouse kappa light chain constant region. The heavy and light chain PCR products were cloned into Sap I linearized antibody vectors containing the IgG1 heavy chain constant region and the kappa light chain constant region, respectively. The heavy chain plasmid has lox2272 and lox511 sites flanking the heavy chain expression cassette. In addition, immediately downstream of lox2272 in the heavy chain plasmid, there is a hygromycin resistance gene lacking a promoter and an initiating ATG. The hygromycin resistance gene is also transcriptionally linked to the downstream eGFP through the IRES sequence. The light chain plasmid has loxP sites and lox2272 sites flanking the light chain expression cassette. In addition, the light chain plasmid has the SV40 promoter immediately preceding the ATG at lox2272, so that upon integration into an appropriate host cell, the SV40 promoter and the initial ATG adjacent to lox2272 in the light chain plasmid read correctly. The frame is adjacent to the hygromycin resistance gene in the heavy chain plasmid, enabling transcription and translation of the hygromycin resistance and eGFP genes. Purified recombinant plasmids with heavy chain variable region sequences were then mixed with plasmids with light chain variable region sequences from the same B cells and transfected together with a plasmid expressing Cre recombinase into a modified CHO host cell line. The improved CHO host cell line comprises from 5' to 3' a loxP site, eCFP, lox2272 site, DsRed and a lox511 site at a transcriptionally active locus. Thus, the host CHO cells can be separated into blue positive, red positive, and green negative cells by flow cytometry. When recombinant plasmids expressing heavy and light chain genes are transfected together with plasmids expressing Cre recombinase, site-specific recombination mediated by Cre recombinase results in integration of the antibody plasmid at a chromosomal locus containing lox sites, resulting in Replacement of eCFP and DsRed genes. The recombinants can then be separated by flow cytometry into blue negative, red negative, green positive cells. Thus, CHO cells transfected with a recombinant plasmid with the heavy chain variable region sequence and a plasmid with the light chain variable region sequence from the same B cells were sorted by flow cytometry and the separation showed blue negative, red negative and green positive phenotype of the correct recombinant, from the isolated clone to establish a stable recombinant CHO cell line expressing the antibody.

Example 3. Determination of antigen binding affinity

 2000或 3000测量抗体对人IL-6R的亲和力。抗体被捕获在抗小鼠IgG表面上，与多种浓度的单体或二聚体形式的重组hIL-6R蛋白相接触。用BIAevaluation^TM软件进行动力学分析，得到结合和解离速率常数。The _KD of antigen binding to the above selected antibodies was determined by measuring their surface kinetics using a real-time biosensor surface plasmon resonance assay (BIAcore ^™ ). More specifically, use

2000 or 3000 measures antibody affinity for human IL-6R. Antibodies were captured on an anti-mouse IgG surface and contacted with various concentrations of recombinant hIL-6R protein in monomeric or dimeric form. Kinetic analysis was performed using BIAevaluation ^™ software to obtain association and dissociation rate constants.

Binding affinity of antibodies to hIL-6R was also determined by plate-based competition immunoassays in the form of hybridoma-conditioned media or purified protein. Antibody protein was purified by protein G affinity chromatography from the conditioned medium of hybridoma cells (Invitrogen) from which bovine IgG had been removed. For the competition ELISA, in brief, different levels of quantified antibodies, premixed with serial dilutions of the antigenic protein hIL-6R-hFc ranging from 0 to 10 μg/ml, were incubated at room temperature for two hours to achieve a cross-talk between antibody and antigen. Pseudobinding equilibrium state. These solutions were then pipetted into 96-well hlL-6R-hFc pre-coated plates, allowing free antibodies in the mixture to bind to hlL-6R-hFc coated on the plates. Plates are typically coated with 1 to 2 μg/ml hIL-6R-hFc protein in PBS, overnight at 4°C, followed by BSA non-specific blocking. After washing excess antibody from the solution, antibody bound to the plate is detected with HRP-conjugated goat anti-mouse IgG or IgA polyclonal antibody reagent and developed with a colorimetric or chemiluminescent substrate. The dependence of the signal on the concentration of antigen in solution was analyzed by 4-parameter fit analysis using Prism ^™ software (Graph Pad Inc.), and reported as _IC50 . Competitive immunoassays were performed using a steady state solution phase Kinexa( ^TM) instrument (Sapidyne Corporation).

The results are shown in Table 1 (control: humanized monoclonal antibody to human IL-6R (US Pat. No. 5,817,790, SEQ ID NO: 69 and 71). Antibody (HCVR and LCVR amino acid sequences): VQ8A9-6 (3, 11); VQ8F11-21 (19, 27); VV7G4-1 (35, 43); VV7G4-10 (51, 59) VV6C10-1 (67, 75); VV6C10-3 (83, 91); VV6C10-4 (99, 107); VV6F12-11 (115, 123); VV9A6-11 (131, 139); VV6A9-5 (147, 155), VV3D8-4 (163, 171); VV1G4-7 (179, 187) ;248982-13-1-E5(195,203);248982-13-2-A9(211,219).The K _D of monomer and dimer is determined by BIAcore ^TM ;The solution K _D is determined by Kinexa ^TM ;IC ₅₀ was determined by ELISA (nd = not determined).

Table 1. Antigen Binding Affinities

Antibody K _D monomer (nM) _KD dimer (nM) Solution K _D monomer (nM) ELISA IC ₅₀ dimer (nM) VQ8A9-6 0.222 0.101 0.120 0.004 VQ8F11-21 0.067 0.023 0.009 0.008 VV3D8-4 2.410 0.172 1.910 0.013 VV6A9-5 0.097 0.146 0.032 0.005 VV1G4-7 0.225 0.070 0.197 0.041 VV6C10-1 0.267 0.032 2.050 0.010 VV6F12-11 nd nd nd 0.033 VV7G4-10 nd nd nd 1.880 VV9A6-11 nd nd nd 0.347 VV6C10-3 nd nd nd 0.009 248982-13-1-E5 0.987 0.785 nd 0.3860 248982-13-2-A9 2.870 nd nd 0.054 control 1.790 nd 1.960 nd

Example 4. Neutralization of hIL-6 activity

Anti-hIL-6R antibodies of the invention were screened for hIL-6 blocking activity using hIL-6 blocking immunoassay, in vitro hIL-6 dependent cell growth bioassay and surface plasmon resonance (BIAcore ^™ ). Immunoassays were used to screen experimental antibodies for their ability to block the binding of hIL-6 to hIL-6R, and in vitro bioassays were used to determine the ability of antibodies to neutralize hIL-6R-mediated cell signaling.

For immunoassays, 96-well plates were coated with hIL-6 recombinant protein in PBS buffer overnight at 4°C. The plate is used to capture free hIL-6R-hFc in the antibody sample solution, and the captured hIL-6R-hFc is quantified according to the standard curve. The sample solution consisted of quantitative hIL-6R-hFc recombinant protein (100pM) and variable antibody, which was serially diluted in the range of 0 to about 50nM in the stock solution of hybridoma conditioned medium or purified antibody protein. Antibody-antigen was incubated at room temperature for ~2 hours to allow antibody-antigen binding to reach equilibrium. The equilibrated sample solutions were then pipetted onto hIL-6 coated plates and free hIL-6R-hFc was measured. After 1 hour of binding, plates were washed and bound hIL-6R-hFc was detected with HRP-conjugated goat anti-hFc polyclonal antibody (Jackson Immuno Research) and developed with TMB substrate (BD Pharmigen). _IC50 was determined as the amount of antibody required to reduce 50% of plate-bound hIL-6 ligand detectable IL-6R-hFc. The results are shown in the first column of Table 2.

Additionally, the ability of the experimental antibodies to block hIL-6 binding to the hIL-6R receptor was determined using surface plasmon resonance. The purified antigen hIL-6R-hFc molecule was captured by goat anti-human IgG polyclonal antibody, which was immobilized on the surface of CM-5 by amine coupling at a density of 250RU. A solution of hIL-6 (0.25ml, 5OnM) was injected onto the receptor surface and bound hIL-6 was recorded (IL-6 first injection). Bound hIL-6 was removed by pulse with 3M MgCl2 _and conditioning buffer. Anti-hlL6R antibody in hybridoma conditioned media was injected onto the captured receptor surface followed by a second injection of hIL-6. The percent reduction in hIL-6 binding due to formation of antibody and receptor complexes was used as a score to define hIL-6 blocking agents, as distinguished from non-blocking agents (second column, Table 2).

Table 2. Neutralization of hIL-6 binding

Antibody hIL6R/hIL6 binding inhibition IC ₅₀ (nM) Inhibition of hIL6/hIL6R binding (%) XG-1 cell proliferation inhibition IC ₅₀ (nM) HepG2/Stat3 luciferase activity IC ₅₀ (nM) VQ8A9-6 0.39 68 0.40 0.097 VQ8F11-21 0.12 98 0.62 0.135 VV3D8-4 0.61 93 >100 nd VV6A9-5 0.35 100 1.1D 0.188 VV1G4-7 1.10 34 1.80 0.578 VV6C10-1 4.60 61 >8.90 nd VV6F12-11 2.20 nd nd nd VV7G4-10 13.00 nd nd nd VV9A6-11 0.50 nd nd nd VV6C10-3 0.06 nd nd nd control 2.20 91 1.50 0.854

The ability of hIL-6R antibodies to block hIL-6 activity in vitro was measured with the hIL-6 dependent myeloma cell line XG-1. XG-1 cells maintained in hIL-6-containing medium were washed twice with hIL-6-free medium, and cultured in hIL-6-free medium for 24 hours to remove residual hIL-6. Starved cells were then pelleted by centrifugation, resuspended in culture medium at 4 x 10 ⁵ cells/ml, and plated in 96-well tissue culture plates at 20,000 cells/well. Purified antibody proteins are serially diluted in culture medium and added to plated cells at concentrations ranging from 0 to 50 nM. Next, recombinant hIL-6 was added to the wells to a final concentration of 8 pM. Cells were grown for ~72 hours at 37°C in a humidified 5% _CO2 incubator. At the end of the growth period, viable cells were measured with a CCK-8 kit (Dojindo, Japan). _IC50s were determined as described above and are reported in the third column of Table 2.

The ability of hIL-6R antibodies to block hIL-6 activity in vitro was also measured in the hIL-6 responsive human hepatoma cell line HepG2. HepG2 cells were transfected with a reporter plasmid containing a STAT3 (signal transducer and activator of transcription 3) response element linked to a luciferase gene. The transfected cells were treated with trypsin, centrifuged, resuspended in culture medium at about 2.5×10 ⁵ cells/ml, and plated in 96-well tissue culture plates with 20,000 cells/well. Purified antibody proteins are serially diluted in culture medium and added to plated cells at concentrations ranging from 0 to 100 nM. Next, recombinant hIL-6 was added to the wells to a final concentration of 50 pM. Responses were measured after incubating cells for 6 h at 37 °C in a humidified 5% _CO2 incubator. Luciferase activity was measured with the Steady-Glo ^™ Luciferase Assay System (Promega). _IC50s were determined as described above and are reported in the fourth column of Table 2.

Example 5. Binding epitope diversity

A competition immunoassay for antibody binding was performed with a control humanized antibody to human IL-6R. Briefly, 96-well immunosorbent plates were coated with 20 ng/well hIL-6R recombinant protein overnight at 4°C. After blocking non-specific binding with BSA, a control antibody was bound to saturate the binding sites of hIL-6R by adding the control at 500 ng/well in half of the plate, and only binding buffer was added in the other half of the plate. After 3 hours of binding at room temperature, purified antibodies were added to a final concentration of 50 ng/ml in the presence or absence of existing control antibodies in the wells. After 1 hour of additional binding, free antibody was washed away, bound antibody was detected on the plate with HRP-conjugated goat anti-mouse IgG or IgA polyclonal antibody, the plate was developed with chromogenic HRP substrate, and absorbance at 450 nm was recorded. The percentage reduction in anti-hlL6R antibody binding due to the presence of the control antibody is listed in Table 3 below. Similar experiments were performed with the surface plasmon resonance technique (Table 3). Both methods give consistent results. The epitopes bound by antibodies VQ8F11, VV3D8, VV6A9, VV6C10-1 overlapped with those of the control antibody; while antibodies VQ8A9, VV1G4, VV6F12, VV7G4, VV9A6, and VV6C10-3 showed binding to different epitopes because the antigen binding was not blocked by the control antibody. Even though the epitopes may not overlap, steric hindrance due to primary antibody binding may result in partial competition.

Table 3. Competition with Control Antibodies for Antigen Binding

Antibody BIAcore ^TM (% reduction) Reduction Determination (% Reduction) VQ8A9-6 26 3 VQ8F11-21 96 79 VV3D8-4 97 84 VV6A9-5 96 84 VV1G4-7 12 3 VV6C10-1 90 80 VV6F12-11 nd 3 VV7G4-10 nd 26 VV9A6-11 nd 18 VV6C10-3 nd 1

Example 6. Species cross-binding properties

The four antibodies were tested for cross-reactivity to monkey IL-6R recombinant protein using BIAcore ^™ technology. Briefly, anti-hIL-6R monoclonal antibodies were presented using a biosensor chip on which goat anti-mouse Fc polyclonal antibodies were immobilized at a density of approximately 75 RU. Recombinant human or monkey monomeric IL-6R protein (Macaca fascicularis, extracellular domain; SEQ ID NO: 251 ) was injected onto the antibody surface at concentrations ranging from 1.25-40 nM. Real-time monitoring of receptor-antibody binding and dissociation of the bound complex. The association rate constant (ka) and dissociation rate constant (kd) were obtained and _KD was calculated (Table 4).

Table 4. Comparison of binding affinity to human IL-6R and monkey IL-6R

Among the four experimental antibodies, VQ8F11, VV6A9, and VQ8A9 strongly reacted with monkey receptors, with K ₀ values reaching about 1.5 to about 3 times that of human receptors, respectively. VV1G4 not blocked by the control antibody (Table 3), despite binding strongly to the human receptor with a _KD of 241 pM, showed no binding to the monkey receptor.

Example 7. Effect of constant regions on binding affinity

Binding of four antibodies with mouse IgG, human IgG1 or human IgG4 (wild type and modified) to monomeric hIL-6R was determined using BIAcore ^™ as described above, except for surface capture of hIgG antibodies using goat anti-human Fc polyclonal antibodies affinity. Monomeric hIL-6R was injected at concentrations of 12.5, 6.25, 3.12 and 1.56 nM. The ability of antibodies to neutralize hIL-6-dependent HepG2/STAT3 signaling ( _IC50 ) was determined using a luciferase assay. The _IC50s of the different IgG isotypes are similar, indicating that isotype has no effect on the affinity of the antibody for the antigen.

Table 5. Comparison of IgG isotypes

Claims (12)

1. antibody or its Fab; It combines with the human interleukin-6 receptor-specific; Wherein said antibody or its Fab comprise the heavy chain CDR1 structural domain shown in the SEQ ID NO:21, the heavy chain CDR2 structural domain shown in the SEQ ID NO:23, the heavy chain CDR3 structural domain shown in the SEQ ID NO:25; Light chain CDR1 structural domain shown in the SEQ ID NO:29, the light chain CDR3 structural domain shown in light chain CDR2 structural domain shown in the SEQ IDNO:31 and the SEQ ID NO:33.

2. the antibody of claim 1 or its Fab comprise the variable region of heavy chain with SEQ ID NO:19.

3. the antibody of claim 1 or its Fab comprise the variable region of light chain with SEQ ID NO:27.

4. each antibody or its Fab among the claim 1-3 comprise variable region of heavy chain with SEQ IDNO:19 and the variable region of light chain with SEQ ID NO:27.

5. each antibody or its Fab among the claim 1-3, wherein said Fab are selected from Fab, F (ab ') ₂And scFv.

6. isolated nucleic acid molecule, its encode each described antibody of aforementioned claim or its Fab.

7. the expression vector that comprises the nucleic acid molecule of claim 6.

8. isolating host cell, it comprises the expression vector of claim 7.

9. the host cell of claim 8, wherein said host cell is intestinal bacteria (E.coli) cell or Chinese hamster ovary celI.

10. the method for preparing anti-IL-8-6 receptor antibody or its Fab comprises and cultivates claim 8 or 9 described host cells, and reclaims antibody or the antibody fragment that is produced.

11. each described antibody of claim 1 to 5 or its Fab are used for alleviating or suppress the purposes of medicine of human diseases or the illness of IL-6 mediation in preparation.

12. the described purposes of claim 11, wherein the disease or the illness of IL-6 mediation are selected from rheumatoid arthritis, inflammatory bowel and systemic lupus erythematous.